BIOCHEMICAL
Vol. 86, No. 2, 1979 January
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 407-414
30, 1979
INTRODUCTION
OF
METHYL INTO
GROUPS
THE
Babiker
Ohio
24,
Gary
E.
of
Biochemistry
State
University
Columbus, November
POOL
and
Department
Received
F -N-METHYLLYSLNE
ONE-CARBON
ElAmin
The
FROM
Ohio
*
Means
43 2 10
1978
methyland c- N-r 3H]Summary: Radioactivity from ingested f - N - hl dimethyllysine residues of reductively methylated casein are rapidly and efficiently absorbed and incorporated into tissues of the chicken. Introduction of label into choline, carnitine and methionine suggests that these amino acids contribute methyl groups to the one-carbon pool.
< - N-Methyl, in
small
common
amounts in
acids
catabolism catalyze di-
and
tissues
and
in
variety
biological
Kakimoto amino
dimethyl
and in
methylation
trimethyllysine
systems Akazawa
human
of proteins
have
many also
urine
Iysine
residues,
have
been
detected
(l-4)
and
appear
to
foods
and
animal
feeds.
detected
and
Protein
proteins. of
residues
including (5)
“normal”
of tissue the
a wide
trimethyllysine
small
suggested
methylase
amounts
that III,
fairly
of these
they
an
be
arose
enzyme
from which
can
residues
of proteins
successively
to
has
demonstrated
in
rat
been
several
the
mono-,
(6).
1: This work is taken from to the Graduate School of the the requirements for the Ph. Acknowledgement is made to administered by the American search. A preliminary account the 174th National Meeting of No 46, Division of Biological
the thesis Ohio State D. degree. the Donors Chemical of some the American Chemistry)
of Babiker University
ElAmin in partial
to
be submitted fulfillment
of The Petroleum Research Fund Society, for support of this reof these results was presented at (Abstract Chemical Society. Chicago, Ill. Aug 29, 1977.
0006-291X/79/020407-08$01.00/0 A07
of
Vol.
86,
No.
2, 1979
BIOCHEMICAL
F- N - Trimethytlysine precursor
of the
and
in
rats
and
thus
Paik
may
be
required
important
(7,
example,
has
8).
In
rats,
for
derivative
of the
and
dimethyllysine
under to
give
number
of
metabolic
version
of
both
has
been
been
shown
The
reduce
metabolic
amino
acids
evidence
just
when
E - N -methyt in
the
report
and
absence
the
suggest
carbon
amino poo
lysine
dimethyllysine
of adequate a further
acids
appear
1 compounds.
enzyme
(11,
12)
and/or
dietary
diets to Their
be
which
of
compounds
as
presence
408
into in
the
lysines.
that
Limiting. a source
C-N-
described
methyl several may
Similarly of
e -N-methyl-
diet
tysine(l3,
biosynthesis
results
Thus
incorporated
for
significance
suggests
for
chickens.
rats
have
substituted
are
importance
the
Lysine in
this
and groups
important therefore
con-
“formatdehyde”
nutritional
methionine
The
catalyze
and
of
utilized
further
a substantial
can
these
carnitine
lysine.
gave
undergo
lysine
for
be
corresponding
Methyl-
undergo
and
asialo
the
to
however,
might
the
appear
E -N-methyl
utilized
for
following
carnitine.
possible
the
have,
asialo-fetuin
requirements
like
be
liver
proteins
containing,
to
to the
nutritional in
An
summarized, might
dietary
F -N-dimethyllysine these
diet
rat
(10)
conditions,
not
apparently
methylated
of tissue
of
do
dietary
dietary
in
in
crassa
directly
et al.
same
do
tissues
directed
Neurospora
catabolism
dimethyllysine
normal
not
no detectable
but
been
trimethyllysine particularly
but conditions
rat
is
derivative
and
the has
minor
the
transformations.
in
both
biosynthetic
1 - trimethyllysine
dimethyllysine
E - N -methyl-
to
the
COMMUNICATIONS
efficient
in
carnitine
Under
those
an
LaBadie
N - r3H
trimethyllysine
attention
many
e-
products
characterized
Little of
and labeled
that
of
fetuin.
unidentified
methylation
an
be
lysine
biosynthesis.
protein,
f - N - [ 3H 1 -methylmany
free
formation
of
to
RESEARCH
carnitine
suggested
a rapid
degradation
shown
however,
carnitine
observed
lysosomal
been
BIOPHYSICAL
metabolite,
(9) have
et al.
AND
from onehave
also 14).
Vol.
86,
No.
important
nutritional
one-carbon residues
sidues
(15)
Materials
Relatively of
may, their
and
AND
consequences
donors.
lysine
supplement
BIOCHEMICAL
2, 1979
dietary in
some
contents
especially simple
proteins cases, of
BIOPHYSICAL
to
in
chemical
the
an
nutritionally
economically available
COMMUNICATIONS
absence
of
other
to
convert
procedures
e -N-methyl-
afford
RESEARCH
and
some
dimethyllysine feasible
one-carbon
reway
to
units.
Methods
Labeling of Protein. Radioactive (methyl - c3H]) mono-and dimethytlysine residues were introduced into 100 mg of vitamin-free casein (Nutritional Biochemicals) by a previously described procedure (15, 16) using 0. 58 mg of ~310 mCi/mmol NaB3H4 (New England Nuclear). Amino acid analysis, using the short column of a Beckman Model 116 analyzer, indicated approximately 20% conversion of lysine to F -N-methyllysine and approximately 30% conversion to F-N-dimethyllysine. A 460 gm, 10 week old broiler Introduction and Uptake of Labeled Protein. type chicken was deprived of food for 12 hrs. A 30 mg sample of the described methyllysine containing casein (-12 u Ci) in 3 ml of 0. 9$NaCl was introduced directly into the crop using a 150 x 2 mm length of stiff polypropylene tubing attached to a hypodermic syringe. A second hypodermic syringe with a needle was used to take 0. 5 ml samples of blood from the wing vein at 30 min intervals. After clotting, lOO,[ samples of the clear amber serum were subjected to liquid scintillation counting in 0. 5 ml of NCS (Amersham) plus 10 ml omnifluor (New England Nuclear) the bird was sacrificed by decapitation after 5;5 hrs and tissue samples were taken and placed on ice or frozen for later characterization. Fractionation of Liver. Samples of fresh or frozen liver of approximately 6. 5 g were homogenized in 10 ml of cold double distilled, deionized water with an ice-cold, motor driven teflon pestle. The crude homogenate was diluted to 30 ml with more water, combined with an equal volume of cold lO$trichloroacetic acid (TCA) and centrifuged for 10 min at -12,000 x g. The solid residue was recovered and reextracted with 20 ml of cold 5%TCA, recentrifuged and the two supernatant fluids were combined and evaporated. The resulting residue was suspended in 4 ml of 2 M HCL, heated vigorously for 2 hrs on a steam bath, cooled and then extracted three times with approximately equal volumes of ether . The remaining sample was again evaporated in vacua and the oily re-sidue was dissolved in 50 ml of 0. 11 M trisodium citrate buffer, pH 5. 25. The TCA insoluble material obtained above was extracted three times with ether and the residue was freeze dried and subjected to hydrolysis in 6 M HCL at 110’ for 22 hrs. After evaporation to dryness in vacua, the sample was dissolved in 0. 11 M trisodium citrate buffer, pH 5. 25. The ether soluble fractions were combined and evaporated on a steam bath. The oily residue was dissolved in 6 ml of 95% ethanol, brought to boil on a steam bath and 6 ml of 2 M HCl were added. After 1 hr of gentle boiling, the sample was cooled, extracted with an equal volume of ether and evaporated to dryness The residue was redissolved in 1 ml of water, extracted --in vacua. twice with 10 ml of ether and again evaporated to dryness. The residue was dissolved in 5 ml of pH 5. 25 citrate buffer.
409
Vol.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Analyses. __ Hydrolyzed liver fractions in 0. 5 ml of pH 5. 25 -Chromatographic _.__.citrate buffer were chromatographed on a 0. 9 x 5 cm column of Beckman spherical cation-exchange resin type PA-35 at 55’ with pH 5. 25 citrate buffer (137. 3 gm trisodium citrate, 26. 2 ml cont. HCl and 0.4 ml caprylic acid per 4.0 1) at a flow rate of 1.0 ml/min. The effluent was monitored continuously for ninhydrin positive material using the standard Beckman Model 116 amino acid analyzer system. Fractions were collected prior to mixing with ninhydrin by use of an 8:1 stream splitter. Radioactivities determined after mixing with 10 ml of Aquasol a Beckman LS-133 liquid scintillation counter. nary ammonium compounds were detected by cipitate upon mixing equal volumes of sample Results
and
--in
methylation vitro
present
to
case
residues
30 min
showed with
only
based
on the
than
70%
Radioactivity and
ether
6%
of the
soluble
radioactive
upon 30 ml,
5OW-X12, radioactive
as
in
in
the
cation
liver
was
after
in soluble
as
shown
sample
recently
described again
to the
upon
estimate that
better
insoluble
to
approximately
contain
two
only
peaks
approximately
4 and carnitine
chromatography
on
Bio-Rad
AG
g ave
results
410
similar authentic
carnitine
two
were
of
with
elution
I)
volumes
(17), eluting
An
with
1, at
later
(Table
TCA
fraction,
chromatography identical
label
soluble,
Figure
hrs
hrs.
appeared in
5;
contents.
5;
and 0. lZpmoles/ml
suggested
TCA
hydrolysis
Thus, exchange
of the
after
present
serum
taken
tissues
the
dimethyllysine
of about
gut
susceptiin
in
tissues
and
retained
Th e ether
A similar
components
feces various
was
liver,
and
distribution
the
its
nevertheless
detected
of major
of the
fractions,
respectively,
choline.
total
label
the
decrease
]-methyl-
rapidly
uniform
amount
components.
observed 29 to
relatively
in
label
were
to
(15),
- r3H
Samples
ingested
shown
to a concentration
ingestion.
relative
been trypsin
F -N
sample
6% of the
of the
bovine
corresponding
and
about
has
from
described
after
a wide
casein by
radioactivity
a maximum
only
and
of
hydrolysis
of the
reached
were using quater preacid (17)
Discussion
Reductive bility
in 0. 5 ml samples (New England Nuclear) Fractions containing appearance of a yellow and 2% phosphomolybdic
with and
the
two
choline
Vol.
86,
No.
Table I. dimethyllysine
2, 1979
BIOCHEMICAL
Tissue in
Distribution the Chicken
of
AND
PH1
BIOPHYSICAL
-label
from
RESEARCH
Ingested
COMMUNICATIONS
pH]-methyl-and
-~-~_-Tissue
Adipose
CPM/g
(Mesenteric
t retroperitoneal)
( x 10-3)a
111
Brain
51
Heart
58
Kidney
112
Liver
Muscle
72
(breast)
85 48
Spleen
a
As determined in 0. 5 ml of
by NCS
liquid plus
10
scintillation ml of
counting omnifluor
BOO-
in
of homogenized toluene as described
tissues in
the
text.
Choline I .
700
-
600-
\.
mo2 v aoo-
1. ‘. 300
-
ILI.
Cornitine
200
\
100
\
1
10
Figure 1. Cation Exchange Fraction. The radioactivity from the acid hydrolyzed, upon chromatography on
:,
!,#d
20 30 Fraction No.
40
50
Chromatography of the Hydrolyzed Ether-Soluble in counts per min. of 1 ml fractions obtained ether-soluble fraction from 640 mg of wet liver type PA-35 resin as described in the text.
411
Vol.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
Ir
RESEARCH
COMMUNICATIONS
I
20
30
i
L
A0
50
60
80
70
Fraction
90
100
110
120
‘0
130
No.
Figure 2. Cation Exchange Chromatography of the Hydrolzyed TCA-Insoluble Fraction. An elution profile for the acid hydrolyzed, TCA-insoluble fraction from 64 mg, wet weight, of liver obtained with a Beckman Model 116 Amino Acid Analyzer is shown in terms of the 570 nm absorbance after reaction with ninhydrin (solid line) and from counts per min. in 1 ml fractions (dash line) as described in the text.
The
TCA
insoluble
in
the
liver.
of
only
as
methionine*(Figure
Hydrolysis
one
Most soluble
major
compounds
contain peaks
respectively in
amino
approximately
acid
analysis
component
radioactivity which
largest
choline,
and radioactive
fraction to
contained
35% revealed
which
eluted
of
the
the in
label
presence
the
same
present
in
volume
2).
of the
appeared three
fraction
smaller
in after
and peaks
(i.
hydrolysis
a number eluted
liver
of at
same in
have
N 6Of)
and
cation
radioactive
the
were
e.
exchange
components volumes
ratios not
was
as
been
carnitine,
1. The degradation of a [14C I - methyllysine cine elastase by rat Liver has recently been amounts of labeled methionine (19).
412
identified containing reported
3).
those
to
The
betaine 5~3~6.
but
TCA
chromatography (Figure
of approximately
the
and Other
at
derivative give rise
31 and
to
of porsmall
BIOCHEMICAL
Vol. 86, No. 2, 1979
AND BIOPHYXAL
RESEARCH COMMUNICATIONS
* Choline
BOOCarnitine
400.,Betaine 300. 0 200s
.
8J1L 10
20
-
30 Fraction
40 No.
50
60
70
Figure 3. Cation Exchange Chromatography of the Hydrolyzed TCA-Soluble Radioactivity in 1 ml fractions obtained from the acid hydrolyzed, Fraction. TCA-soluble fraction from 64 mg, wet weight of liver upon chromatography on type PA-35 resin as described in the text.
36 ml
eluted
at the
methyllysine, The in
the
and
volumes
results
described
domestic
above
chicken
pool case
methylated factors
and amino
including
mammalian choline
methionine:
for
compounds. the acid
species,
authentic
in
species
dimethyl-
thought
of
and
to
for
liver
have very
phosphatidylethanolamine
mono-
synthesis
several in
these
reflect
a variety
for
example,
unlike
dietary
requirement
levels
the
same
may
a specific low
of
carnitine
from
(10)
of pathways
~-N-methyl-
of
formation
methyltransferase
413
dietary the
Thus,
reflect
existence
formation
its
rat
the of
utilized
difference. chickens
indicate
groups
apparent
lack
residues
young is
are The
apparent
to
methyl
residues
the
which
appear
whereby
E -N-dimethyllysine
present
observed
respectively.
one-carbon
for
same
of most
of S-adenosylactivity
(20,
21).
Vol.
86,
The
No.
rapid
the
absorption
existence
amino
be
nutritionally
of
one-carbon
ways will
and be
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
the
have
salvage
been
one-carbon
possible reported
for Additional
nutritional in
detail
BIOPHYSICAL
only
examined
the
of
in
(22),
suggests
into
for
these
studies
their
rapid
tissue
the above
dietary in
and
as
the
suggests
minor
the
efficient
presence
characterize support
COMMUNICATIONS
amount
presumably to
consequences
small this
that
chicken,
RESEARCH
label
mechanism
present
pool
significant units.
AND
incorporation
Although
which
into
high
efficient
residues.
proteins
poration
and
of an
acid
tary
BIOCHEMICAL
2, 1979
in an
few
dieincor-
the
diet
alternate
metabolic results
may source
pathand
later.
Murray., K. (1964) Biochemistry_3, 10-15. Paik, W. K. and Kim, S. (1967) Biochem. Biophys. Res. Commun. 27, 479-483. DeLange, R. J., Glazer, A. N. and Smith, E. L., (1969) J. Biol. Chem. 1385-1388. 244, Huszer, G., (1972), J. Bio!. Cl-lz:n. 247, 4057-4062. Kakimoto, Y. and Akazawa, S. (1970) 7..Biol. Chem. 5751-5758. -’245 and Kim, S. (1970) J. Biol. Chem. 245, 6010-6015. Paik, W. K., Horne, D. W. , and Broquist, H. P. (1973) J. Biol. Chem. 248, 2170-2175, Tanphaichitr,, V., and Broquist, H. P. (1973) J. Biol. Chem. 248, 21762181. Paik, W. K., Nochumson, S. and Kim. S. (1977) Trends Biochem. Sci. 2, 159-161,. GBadie, J. H, ., Dunn, W. A. and Aronson, N. N. (1976) Biochem. J. 85-95. 160, Kim, S., Benoiton, J. , and Paik, W. K. (1964) J. Biol. Chem. 239, 3790-3796. Paik, W. K. and Kim. S. (1974) Arch. Biochem. Biophys. 165, 369-378. (1944) Biochem. J. 38, 125 -129. Neuberger, A. and Sanger, F. Lee, H. S., Sen, L. C., Clifford, A. J., Whitaker, J. R. ard Feeney, R. E. (1978) J. Nutr. 108, 687-697. Means G. E. and Feeney, R. E., (1968) Biochemistry L, 2192-2201. Lin, Y., Means, G. E., and Feeney, R. E. (1969) J. Biol. Chem. 24, 789-793. ElAmin, B., (1977) M. S. Thesis, The Ohio State University, Columbus, Ohio. p. 13. Hulse, J. D., Ellis, S. R., and Henderson, L. M. (1978) J. Biol. Chem. 253, 1654-1659. Gan, J. C. and Fretz, J. C. (1978) Fed. Proc. 37, 1811 (abstract no 2964. Jukes, T. H., Oleson, J. J., and Dornbush, A.?. (1945) J. Nutr. 30, 219-223. Molitoris, B. A. and Baker, D. H. (1976), J. Nutr. lx, 412-418. Matsuoka, M. (1972) Seikagaku 42, 364 as quoted by Paik, W. K. and Kim, S. (1975) Advances in Enzymology 42, 227-286.
414
Vol. 86, No. 2, 1979 January
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 407-414
30, 1979
INTRODUCTION
OF
METHYL INTO
GROUPS
THE
Babiker
Ohio
24,
Gary
E.
of
Biochemistry
State
University
Columbus, November
POOL
and
Department
Received
F -N-METHYLLYSLNE
ONE-CARBON
ElAmin
The
FROM
Ohio
*
Means
43 2 10
1978
methyland c- N-r 3H]Summary: Radioactivity from ingested f - N - hl dimethyllysine residues of reductively methylated casein are rapidly and efficiently absorbed and incorporated into tissues of the chicken. Introduction of label into choline, carnitine and methionine suggests that these amino acids contribute methyl groups to the one-carbon pool.
< - N-Methyl, in
small
common
amounts in
acids
catabolism catalyze di-
and
tissues
and
in
variety
biological
Kakimoto amino
dimethyl
and in
methylation
trimethyllysine
systems Akazawa
human
of proteins
have
many also
urine
Iysine
residues,
have
been
detected
(l-4)
and
appear
to
foods
and
animal
feeds.
detected
and
Protein
proteins. of
residues
including (5)
“normal”
of tissue the
a wide
trimethyllysine
small
suggested
methylase
amounts
that III,
fairly
of these
they
an
be
arose
enzyme
from which
can
residues
of proteins
successively
to
has
demonstrated
in
rat
been
several
the
mono-,
(6).
1: This work is taken from to the Graduate School of the the requirements for the Ph. Acknowledgement is made to administered by the American search. A preliminary account the 174th National Meeting of No 46, Division of Biological
the thesis Ohio State D. degree. the Donors Chemical of some the American Chemistry)
of Babiker University
ElAmin in partial
to
be submitted fulfillment
of The Petroleum Research Fund Society, for support of this reof these results was presented at (Abstract Chemical Society. Chicago, Ill. Aug 29, 1977.
0006-291X/79/020407-08$01.00/0 A07
of
Vol.
86,
No.
2, 1979
BIOCHEMICAL
F- N - Trimethytlysine precursor
of the
and
in
rats
and
thus
Paik
may
be
required
important
(7,
example,
has
8).
In
rats,
for
derivative
of the
and
dimethyllysine
under to
give
number
of
metabolic
version
of
both
has
been
been
shown
The
reduce
metabolic
amino
acids
evidence
just
when
E - N -methyt in
the
report
and
absence
the
suggest
carbon
amino poo
lysine
dimethyllysine
of adequate a further
acids
appear
1 compounds.
enzyme
(11,
12)
and/or
dietary
diets to Their
be
which
of
compounds
as
presence
408
into in
the
lysines.
that
Limiting. a source
C-N-
described
methyl several may
Similarly of
e -N-methyl-
diet
tysine(l3,
biosynthesis
results
Thus
incorporated
for
significance
suggests
for
chickens.
rats
have
substituted
are
importance
the
Lysine in
this
and groups
important therefore
con-
“formatdehyde”
nutritional
methionine
The
catalyze
and
of
utilized
further
a substantial
can
these
carnitine
lysine.
gave
undergo
lysine
for
be
corresponding
Methyl-
undergo
and
asialo
the
to
however,
might
the
appear
E -N-methyl
utilized
for
following
carnitine.
possible
the
have,
asialo-fetuin
requirements
like
be
liver
proteins
containing,
to
to the
nutritional in
An
summarized, might
dietary
F -N-dimethyllysine these
diet
rat
(10)
conditions,
not
apparently
methylated
of tissue
of
do
dietary
dietary
in
in
crassa
directly
et al.
same
do
tissues
directed
Neurospora
catabolism
dimethyllysine
normal
not
no detectable
but
been
trimethyllysine particularly
but conditions
rat
is
derivative
and
the has
minor
the
transformations.
in
both
biosynthetic
1 - trimethyllysine
dimethyllysine
E - N -methyl-
to
the
COMMUNICATIONS
efficient
in
carnitine
Under
those
an
LaBadie
N - r3H
trimethyllysine
attention
many
e-
products
characterized
Little of
and labeled
that
of
fetuin.
unidentified
methylation
an
be
lysine
biosynthesis.
protein,
f - N - [ 3H 1 -methylmany
free
formation
of
to
RESEARCH
carnitine
suggested
a rapid
degradation
shown
however,
carnitine
observed
lysosomal
been
BIOPHYSICAL
metabolite,
(9) have
et al.
AND
from onehave
also 14).
Vol.
86,
No.
important
nutritional
one-carbon residues
sidues
(15)
Materials
Relatively of
may, their
and
AND
consequences
donors.
lysine
supplement
BIOCHEMICAL
2, 1979
dietary in
some
contents
especially simple
proteins cases, of
BIOPHYSICAL
to
in
chemical
the
an
nutritionally
economically available
COMMUNICATIONS
absence
of
other
to
convert
procedures
e -N-methyl-
afford
RESEARCH
and
some
dimethyllysine feasible
one-carbon
reway
to
units.
Methods
Labeling of Protein. Radioactive (methyl - c3H]) mono-and dimethytlysine residues were introduced into 100 mg of vitamin-free casein (Nutritional Biochemicals) by a previously described procedure (15, 16) using 0. 58 mg of ~310 mCi/mmol NaB3H4 (New England Nuclear). Amino acid analysis, using the short column of a Beckman Model 116 analyzer, indicated approximately 20% conversion of lysine to F -N-methyllysine and approximately 30% conversion to F-N-dimethyllysine. A 460 gm, 10 week old broiler Introduction and Uptake of Labeled Protein. type chicken was deprived of food for 12 hrs. A 30 mg sample of the described methyllysine containing casein (-12 u Ci) in 3 ml of 0. 9$NaCl was introduced directly into the crop using a 150 x 2 mm length of stiff polypropylene tubing attached to a hypodermic syringe. A second hypodermic syringe with a needle was used to take 0. 5 ml samples of blood from the wing vein at 30 min intervals. After clotting, lOO,[ samples of the clear amber serum were subjected to liquid scintillation counting in 0. 5 ml of NCS (Amersham) plus 10 ml omnifluor (New England Nuclear) the bird was sacrificed by decapitation after 5;5 hrs and tissue samples were taken and placed on ice or frozen for later characterization. Fractionation of Liver. Samples of fresh or frozen liver of approximately 6. 5 g were homogenized in 10 ml of cold double distilled, deionized water with an ice-cold, motor driven teflon pestle. The crude homogenate was diluted to 30 ml with more water, combined with an equal volume of cold lO$trichloroacetic acid (TCA) and centrifuged for 10 min at -12,000 x g. The solid residue was recovered and reextracted with 20 ml of cold 5%TCA, recentrifuged and the two supernatant fluids were combined and evaporated. The resulting residue was suspended in 4 ml of 2 M HCL, heated vigorously for 2 hrs on a steam bath, cooled and then extracted three times with approximately equal volumes of ether . The remaining sample was again evaporated in vacua and the oily re-sidue was dissolved in 50 ml of 0. 11 M trisodium citrate buffer, pH 5. 25. The TCA insoluble material obtained above was extracted three times with ether and the residue was freeze dried and subjected to hydrolysis in 6 M HCL at 110’ for 22 hrs. After evaporation to dryness in vacua, the sample was dissolved in 0. 11 M trisodium citrate buffer, pH 5. 25. The ether soluble fractions were combined and evaporated on a steam bath. The oily residue was dissolved in 6 ml of 95% ethanol, brought to boil on a steam bath and 6 ml of 2 M HCl were added. After 1 hr of gentle boiling, the sample was cooled, extracted with an equal volume of ether and evaporated to dryness The residue was redissolved in 1 ml of water, extracted --in vacua. twice with 10 ml of ether and again evaporated to dryness. The residue was dissolved in 5 ml of pH 5. 25 citrate buffer.
409
Vol.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Analyses. __ Hydrolyzed liver fractions in 0. 5 ml of pH 5. 25 -Chromatographic _.__.citrate buffer were chromatographed on a 0. 9 x 5 cm column of Beckman spherical cation-exchange resin type PA-35 at 55’ with pH 5. 25 citrate buffer (137. 3 gm trisodium citrate, 26. 2 ml cont. HCl and 0.4 ml caprylic acid per 4.0 1) at a flow rate of 1.0 ml/min. The effluent was monitored continuously for ninhydrin positive material using the standard Beckman Model 116 amino acid analyzer system. Fractions were collected prior to mixing with ninhydrin by use of an 8:1 stream splitter. Radioactivities determined after mixing with 10 ml of Aquasol a Beckman LS-133 liquid scintillation counter. nary ammonium compounds were detected by cipitate upon mixing equal volumes of sample Results
and
--in
methylation vitro
present
to
case
residues
30 min
showed with
only
based
on the
than
70%
Radioactivity and
ether
6%
of the
soluble
radioactive
upon 30 ml,
5OW-X12, radioactive
as
in
in
the
cation
liver
was
after
in soluble
as
shown
sample
recently
described again
to the
upon
estimate that
better
insoluble
to
approximately
contain
two
only
peaks
approximately
4 and carnitine
chromatography
on
Bio-Rad
AG
g ave
results
410
similar authentic
carnitine
two
were
of
with
elution
I)
volumes
(17), eluting
An
with
1, at
later
(Table
TCA
fraction,
chromatography identical
label
soluble,
Figure
hrs
hrs.
appeared in
5;
contents.
5;
and 0. lZpmoles/ml
suggested
TCA
hydrolysis
Thus, exchange
of the
after
present
serum
taken
tissues
the
dimethyllysine
of about
gut
susceptiin
in
tissues
and
retained
Th e ether
A similar
components
feces various
was
liver,
and
distribution
the
its
nevertheless
detected
of major
of the
fractions,
respectively,
choline.
total
label
the
decrease
]-methyl-
rapidly
uniform
amount
components.
observed 29 to
relatively
in
label
were
to
(15),
- r3H
Samples
ingested
shown
to a concentration
ingestion.
relative
been trypsin
F -N
sample
6% of the
of the
bovine
corresponding
and
about
has
from
described
after
a wide
casein by
radioactivity
a maximum
only
and
of
hydrolysis
of the
reached
were using quater preacid (17)
Discussion
Reductive bility
in 0. 5 ml samples (New England Nuclear) Fractions containing appearance of a yellow and 2% phosphomolybdic
with and
the
two
choline
Vol.
86,
No.
Table I. dimethyllysine
2, 1979
BIOCHEMICAL
Tissue in
Distribution the Chicken
of
AND
PH1
BIOPHYSICAL
-label
from
RESEARCH
Ingested
COMMUNICATIONS
pH]-methyl-and
-~-~_-Tissue
Adipose
CPM/g
(Mesenteric
t retroperitoneal)
( x 10-3)a
111
Brain
51
Heart
58
Kidney
112
Liver
Muscle
72
(breast)
85 48
Spleen
a
As determined in 0. 5 ml of
by NCS
liquid plus
10
scintillation ml of
counting omnifluor
BOO-
in
of homogenized toluene as described
tissues in
the
text.
Choline I .
700
-
600-
\.
mo2 v aoo-
1. ‘. 300
-
ILI.
Cornitine
200
\
100
\
1
10
Figure 1. Cation Exchange Fraction. The radioactivity from the acid hydrolyzed, upon chromatography on
:,
!,#d
20 30 Fraction No.
40
50
Chromatography of the Hydrolyzed Ether-Soluble in counts per min. of 1 ml fractions obtained ether-soluble fraction from 640 mg of wet liver type PA-35 resin as described in the text.
411
Vol.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
Ir
RESEARCH
COMMUNICATIONS
I
20
30
i
L
A0
50
60
80
70
Fraction
90
100
110
120
‘0
130
No.
Figure 2. Cation Exchange Chromatography of the Hydrolzyed TCA-Insoluble Fraction. An elution profile for the acid hydrolyzed, TCA-insoluble fraction from 64 mg, wet weight, of liver obtained with a Beckman Model 116 Amino Acid Analyzer is shown in terms of the 570 nm absorbance after reaction with ninhydrin (solid line) and from counts per min. in 1 ml fractions (dash line) as described in the text.
The
TCA
insoluble
in
the
liver.
of
only
as
methionine*(Figure
Hydrolysis
one
Most soluble
major
compounds
contain peaks
respectively in
amino
approximately
acid
analysis
component
radioactivity which
largest
choline,
and radioactive
fraction to
contained
35% revealed
which
eluted
of
the
the in
label
presence
the
same
present
in
volume
2).
of the
appeared three
fraction
smaller
in after
and peaks
(i.
hydrolysis
a number eluted
liver
of at
same in
have
N 6Of)
and
cation
radioactive
the
were
e.
exchange
components volumes
ratios not
was
as
been
carnitine,
1. The degradation of a [14C I - methyllysine cine elastase by rat Liver has recently been amounts of labeled methionine (19).
412
identified containing reported
3).
those
to
The
betaine 5~3~6.
but
TCA
chromatography (Figure
of approximately
the
and Other
at
derivative give rise
31 and
to
of porsmall
BIOCHEMICAL
Vol. 86, No. 2, 1979
AND BIOPHYXAL
RESEARCH COMMUNICATIONS
* Choline
BOOCarnitine
400.,Betaine 300. 0 200s
.
8J1L 10
20
-
30 Fraction
40 No.
50
60
70
Figure 3. Cation Exchange Chromatography of the Hydrolyzed TCA-Soluble Radioactivity in 1 ml fractions obtained from the acid hydrolyzed, Fraction. TCA-soluble fraction from 64 mg, wet weight of liver upon chromatography on type PA-35 resin as described in the text.
36 ml
eluted
at the
methyllysine, The in
the
and
volumes
results
described
domestic
above
chicken
pool case
methylated factors
and amino
including
mammalian choline
methionine:
for
compounds. the acid
species,
authentic
in
species
dimethyl-
thought
of
and
to
for
liver
have very
phosphatidylethanolamine
mono-
synthesis
several in
these
reflect
a variety
for
example,
unlike
dietary
requirement
levels
the
same
may
a specific low
of
carnitine
from
(10)
of pathways
~-N-methyl-
of
formation
methyltransferase
413
dietary the
Thus,
reflect
existence
formation
its
rat
the of
utilized
difference. chickens
indicate
groups
apparent
lack
residues
young is
are The
apparent
to
methyl
residues
the
which
appear
whereby
E -N-dimethyllysine
present
observed
respectively.
one-carbon
for
same
of most
of S-adenosylactivity
(20,
21).
Vol.
86,
The
No.
rapid
the
absorption
existence
amino
be
nutritionally
of
one-carbon
ways will
and be
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
the
have
salvage
been
one-carbon
possible reported
for Additional
nutritional in
detail
BIOPHYSICAL
only
examined
the
of
in
(22),
suggests
into
for
these
studies
their
rapid
tissue
the above
dietary in
and
as
the
suggests
minor
the
efficient
presence
characterize support
COMMUNICATIONS
amount
presumably to
consequences
small this
that
chicken,
RESEARCH
label
mechanism
present
pool
significant units.
AND
incorporation
Although
which
into
high
efficient
residues.
proteins
poration
and
of an
acid
tary
BIOCHEMICAL
2, 1979
in an
few
dieincor-
the
diet
alternate
metabolic results
may source
pathand
later.
Murray., K. (1964) Biochemistry_3, 10-15. Paik, W. K. and Kim, S. (1967) Biochem. Biophys. Res. Commun. 27, 479-483. DeLange, R. J., Glazer, A. N. and Smith, E. L., (1969) J. Biol. Chem. 1385-1388. 244, Huszer, G., (1972), J. Bio!. Cl-lz:n. 247, 4057-4062. Kakimoto, Y. and Akazawa, S. (1970) 7..Biol. Chem. 5751-5758. -’245 and Kim, S. (1970) J. Biol. Chem. 245, 6010-6015. Paik, W. K., Horne, D. W. , and Broquist, H. P. (1973) J. Biol. Chem. 248, 2170-2175, Tanphaichitr,, V., and Broquist, H. P. (1973) J. Biol. Chem. 248, 21762181. Paik, W. K., Nochumson, S. and Kim. S. (1977) Trends Biochem. Sci. 2, 159-161,. GBadie, J. H, ., Dunn, W. A. and Aronson, N. N. (1976) Biochem. J. 85-95. 160, Kim, S., Benoiton, J. , and Paik, W. K. (1964) J. Biol. Chem. 239, 3790-3796. Paik, W. K. and Kim. S. (1974) Arch. Biochem. Biophys. 165, 369-378. (1944) Biochem. J. 38, 125 -129. Neuberger, A. and Sanger, F. Lee, H. S., Sen, L. C., Clifford, A. J., Whitaker, J. R. ard Feeney, R. E. (1978) J. Nutr. 108, 687-697. Means G. E. and Feeney, R. E., (1968) Biochemistry L, 2192-2201. Lin, Y., Means, G. E., and Feeney, R. E. (1969) J. Biol. Chem. 24, 789-793. ElAmin, B., (1977) M. S. Thesis, The Ohio State University, Columbus, Ohio. p. 13. Hulse, J. D., Ellis, S. R., and Henderson, L. M. (1978) J. Biol. Chem. 253, 1654-1659. Gan, J. C. and Fretz, J. C. (1978) Fed. Proc. 37, 1811 (abstract no 2964. Jukes, T. H., Oleson, J. J., and Dornbush, A.?. (1945) J. Nutr. 30, 219-223. Molitoris, B. A. and Baker, D. H. (1976), J. Nutr. lx, 412-418. Matsuoka, M. (1972) Seikagaku 42, 364 as quoted by Paik, W. K. and Kim, S. (1975) Advances in Enzymology 42, 227-286.
414
